In support of this idea, data from our laboratory indicate that esophageal cancer cells are dependent on glutamine. We observed significant reduction in both proliferation and invasion of the adenocarcinoma cancer cell lines Flo-1 and Esc2 after withdrawal of glutamine regardless of the presence of glucose. We have shown that lentiviral shRNA blockage of GLS-1, the enzyme that converts glutamine to glutamate for entry into the tricarboxylic acid cycle, mimics the effects of glutamine withdrawal. We have preliminary data suggesting that withdrawal of glutamine induces cellular senescence and autophagy: 1) Using flow cytometry, we found that the cell cycle's G1 phase is increased and its S phase is decreased, indicating a lack of cell division characteristic of senescence; and 2) We observed induction of autophagosomal marker LC3 and no apoptosis. We further explored this mechanism by studying the cellular respiration. On the Seahorse machine (which measures oxygen utilization and acid production by a living cell in real time), glutamine raised the oxygen consumption rate during mitochondrial stress-testing in Flo-1 and Esc2 esophageal cancer cells. However, this response was blunted to different degrees with the addition of glucose or in the GLS-1 knockdown, suggesting a differential induction of the Warburg phenomenon in each line. These cells also had differential increases in extracellular acidification rate with exposure to glucose and glutamine, further supporting differences in the Warburg effect. These differences suggest that tumor-specific metabolic treatment strategies may be required for optimum synergistic cancer cell death and individual patient-directed therapy. Upon withdrawal of glutamine, glycolytic pathway proteins glut1 transporter and hexokinase 2 increase, and the mitochondrial transporter uncoupled protein 2 (UCP2) decrease, further suggesting that targeting the mitochondria rather than the glycolytic pathway may be a valuable treatment strategy. Future experiments will be divided into: 1) Analysis of metabolomic output in vitro and in patient protocols to identify biomarkers of treatment outcomes; 2) Exploration of metabolic pathways appropriate for treatment targets; and 3) Evaluation of cellular autophagy and apoptosis induced by combining metabolic inhibitors with standard therapeutics. To achieve these goals, we will examine the metabolomic output of cells treated without glutamine and GLS-1 shRNA transductions. In addition, we will restore TCA intermediaries such as alpha-ketoglutarate to examine whether the sickly phenotype of glutamine-depleted cells and GLS-1 shRNA lentiviral-treated cancer cells can be reversed to support these pathways as relevant cancer targets. Identification of these metabolites is also part of a biomarker-based, clinical protocol currently under development. To further characterize the oncogenic mechanisms, we are pursuing blockage of UCP2 as a potential therapeutic target. In addition, to identify other currently unknown targets, we plan to assess global gene expression analysis through RNAseq. These experiments would allow us to detect sets of possible targets worth pursuing to achieve individual, patient-centered, precision medicine. Ultimately, based on the most robust oncogenic mechanisms we find, we plan to study the synergistic effects of mitochondrial inhibition and cisplatin to determine whether autophagy, apoptosis, and cytotoxicity can be increased. We are currently working in collaboration with another laboratory to study novel, targeted mitochondrial inhibitors that may translate into translational protocols.